In the Laboratory edited by
Computer Bulletin Board
Steven D. Gammon
Application of a Datalogger in Biosensing: A Reagentless Hydrogen Peroxide Biosensor
Western Washington University Bellingham, WA 98225-9150
W
Lihua Ma and Martin M. F. Choi* Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong, SAR, China; *
[email protected] In an era of unprecedented interest in biochemistry, how to introduce undergraduate and postgraduate students to the biochemistry world is one of the most interesting and challenging topics. To date biosensing technology is one of the hottest research topics. A biosensor is normally defined as a device incorporating a biological molecular recognition component to a transducer that can output an electronic signal proportional to the concentration of the analyte being sensed (1). The high selectivity in biosensors provided by the biological recognition systems including antibodies, cells, enzymes, nucleic acids, and receptors has been used to detect important biological molecules such as amino acids, antigens, creatinine, glucose, nucleic acids, and urea. Enzymes are biological recognition molecules commonly employed in research and development because most chemical reactions in living systems are catalyzed by enzymes. A datalogger, which is a computer interfaced to one or more sensors, is becoming popular in biology, chemistry, and physics teaching laboratories because students can simultaneously monitor, in real time, system parameters, such as temperature, pH, oxygen concentration, and so on. We have reported the application of an oxygen sensor in conjunction with a datalogger to real-time monitor the liberation of dissolved oxygen in the photosynthesis of seaweed (2) and to determine glucose concentration (3). Eggshells from industries that use only the inside of the egg are a significant waste disposal problem. The development of value-added byproducts from this waste, therefore, would be welcomed. An eggshell membrane is a light pink double-layered membrane inside the eggshell composed of highly cross-linked proteins similar to keratin, collagen, and elastin (4), which is a suitable bioplatform for enzyme immobilization (5, 6). It has been reported that eggshell membranes contain small quantities of catalase (7). In nature catalase plays a significant role as a hydrogen peroxide (H2O2) scavenger in protecting cells against the oxidation effect of H2O2 (8, 9). In this article we describe an eggshell membrane in conjunction with an oxygen electrode and a datalogging system employed to fabricate a H2O2 biosensor. To date although there are numerous methods for the determination of H2O2, including fluorimetry (10), amperometry (11–13), and chemiluminometry (14, 15); most of these methods require extensive or laborious chemical reactions and complicated or expensive instrumentation. In this article we present a simple biosensor method for the determination of H2O2. A fresh eggshell membrane was mounted onto an oxygen electrode. The oxygen electrode was then placed in contact with a sample solution for measurement of H2O2. The detection of H2O2 is based on the increase in the dissolved oxygen in the sample solution. When 862
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H2O2 is present in the sample solution, it will be enzymatically decomposed by the catalase in the eggshell membrane with a concomitant increase in the dissolved oxygen concentration in the sample solution (7) 2H2O2
catalase
2H2O + O2
The concentration of H2O2 can be directly determined by the increase in the dissolved oxygen content in the solution. The results compared well with the conventional iodometric titration method of H2O2 (16). The proposed method is simple, safe, and convenient to use. In particular, this method focuses on the development of practical knowledge of biosensors and also fosters understanding of computational methods. Experimental Procedure The experimental setup has been described in a previous article (3). A fresh egg was purchased from a local market. The membrane was carefully peeled from the broken eggshell after the albumen and yolk had been removed. It was cleaned with a copious quantities of deionized water. The membrane was placed in a clean watch glass and it was cut into a circle ca. 1.5 cm in diameter. It was then stored in a pH 6.8 phosphate buffer1 (25 mM) at 4 ⬚C overnight before use.
Figure 1. The dissolved oxygen content of a phosphate buffer (pH 6.8) after each successive addition of 100 µL of H2O2 standard (0.3 M): (a) no H2O2 standard added, (b–e) successive additions of 100 µL of 0.3 M H2O2 standard. The inset displays the linear calibration plot for the biosensor (dissolved oxygen = 2.693 [H2O2] + 0.008; r2 = 0.9991).
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A few drops of pH 6.8 phosphate buffer were put on the surface of a Pasco CI-6542 oxygen sensor.2 An eggshell membrane was carefully positioned on the surface of the oxygen electrode and kept in position by an O-ring. Introduction of air bubbles between the eggshell membrane and oxygen electrode was carefully avoided. The electrode was then immersed into a stirred 50-mL phosphate buffer solution, pH 6.8. Successive 100 µL of H2O2 standard3 (0.3 M) or sample H2O2 solutions were injected into the phosphate buffer with the use of a 0.25-mL syringe. The dissolved oxygen signal was captured at a sampling rate of 10 per second and processed by a datalogger system consisting of a ScienceWorkshop 500 interface, serial cables, a power supply, and control software.2 The data were logged in a notebook PC for real-time display and processing as shown in Figure 1. When the H2O2 biosensor was not in use, the eggshell membrane was removed from the oxygen electrode and stored in a pH 6.8 phosphate buffer (25 mM) at 4 ⬚C.
Figure 2. Effect of phosphate buffer concentration (pH 6.8) on the sensitivity of the biosensor.
Hazards Hydrogen peroxide is irritating and corrosive in high concentrations (> 10%). Students must wear splash goggles, gloves, apron, trousers, and rubber boots to prevent contact. Inhalation of mist will cause irritation of lungs, throat, and nose that usually subsides after exposure has ended. Higher exposures will cause skin burns and ulceration. Concentrated vapor or mist causes eye irritation with discomfort, tearing, blurring of vision, and possibly severe eye injury and blindness. Solutions of 3% or less cause pain but no damage; higher concentrations may cause eye damage with corneal ulceration. Results and Discussion The oxygen electrode acting as an oxygen transducer was employed to measure the rate of oxygen released in the enzymatic decomposition of H2O2. The analytical signal of the H2O2 biosensor is the increase in the dissolved oxygen content upon exposure to H2O2 solution. A typical response curve of the H2O2 biosensor is shown in Figure 1. The increase in oxygen level was found to be proportional to the H2O2 concentration. A linear calibration curve plotting the oxygen level against the concentration of H2O2 is displayed in the inset in Figure 1. The slope of the calibration curve denotes the sensitivity for the H2O2 detection; the biosensor shows good response to H2O2. In addition, there is no increase in dissolved oxygen when H2O2 is added to a solution if the oxygen electrode has not been previously covered with an eggshell membrane. The effect of phosphate buffer concentration on the sensitivity of the biosensor was investigated by subjecting the biosensor to phosphate buffer from 5.0 to 400 mM at pH 6.8 (Figure 2). The sensitivity of the biosensor remained relatively constant throughout the range. Similarly, the effect of ionic strength on the sensitivity of the biosensor was studied by subjecting the biosensor to phosphate buffers (pH 6.8, 25 mM) containing 0–0.4 M KCl (Figure 3). It was found that the response of the eggshell biosensor for H2O2 was almost independent of the ionic strength over a wide range. Furthermore, the pH effect was also studied over the range of 4.5–9.2 (Figure 4). The results showed that the optimal www.JCE.DivCHED.org
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Figure 3. Effect of ionic strength (controlled by KCl) on the sensitivity of the biosensor.
Figure 4. Effect of pH on the sensitivity of the biosensor.
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Supplemental Material
Instructions for the students and notes for the instructor are available in this issue of JCE Online. Acknowledgment Financial support of this work from HKBU (project FRG/01-02/II-08) is gratefully acknowledged. Notes Figure 5. Effect of temperature on the sensitivity of the biosensor.
Table 1. Determination of Hydrogen Peroxide Concentrations in Commercial Products Using the Proposed Hydrogen Peroxide Biosensor and Iodometric Methods Sample No.
Method ᎑1
Proposed/(mol L )
Iodometric/(mol L᎑1)
1
1.98
1.84
2
1.94
1.85
3
2.02
1.93
4
2.06
1.94
pH value was around 6.8 and the sensitivity could be maintained above 90% for the investigated pH range. The pH value has little effect on the dynamic working range of the biosensor. Figure 5 shows the effect of temperature on the sensitivity of the biosensor. The response decreased with the increase in the working temperature and remained relatively constant in the range of 17–30 ⬚C. The sensitivity started to decline sharply above 35 ⬚C. Finally, the H2O2 concentrations of some commercial samples were determined by the H2O2 biosensor. The results compared well with a standard iodometric method (16) and are displayed in Table 1. The use of an eggshell membrane, an oxygen electrode, and a datalogger provide a convenient and simple method for H2O2 determination. The proposed H2O2 biosensor is easy to fabricate and can be used repeatedly for at least several months. With the experiments described here we can also introduce students to the concept of biosensor technology.
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1. Monosodium dihydrogen phosphate and disodium hydrogen phosphate were purchased from Farco Chemical Supplies (Beijing). 2. Pasco CI-6542 oxygen sensor and ScienceWorkshop 500 interface were purchased from Pasco Scientific, Roseville, CA. http:// www.pasco.com (accessed Feb 2004). The total cost is approximately $720. Alternative sources for oxygen probes and their accessories are Vernier Software & Technology, Beaverton, OR, http:// www.vernier.com (accessed Feb 2004) and PP Systems, Haverhill, MA, http://www.ppsystems.com (accessed Feb 2004). 3. Hydrogen peroxide solution 35 % w兾w in water was from Acros Organics (Geel, Belgium).
Literature Cited 1. Leech, D. Chem. Soc. Rev. 1994, 23, 205. 2. Choi, M. M. F.; Wong, P. S.; Yiu, T. P. J. Chem. Educ. 2002, 79, 980. 3. Choi, M. M. F.; Wong, P. S. J. Chem. Educ. 2002, 79, 982. 4. Baker, J. R.; Balch, D. A. Biochem. J. 1962, 82, 352. 5. Choi, M. M. F.; Pang, W. H. S.; Xiao, D.; Wu, X. Analyst 2001, 126, 1558. 6. Xiao, D; Choi, M. M. F. Anal. Chem. 2002, 74, 863. 7. Akagawa, M.; Wako, Y.; Suyama, K. Biochim. Biophys. Acta 1999, 1434, 151. 8. Fridovich, I.; Freeman, B. Annu. Rev. Physiol. 1986, 48, 693. 9. Buckley, B. J.; Tanswell, A. K.; Freeman, B. A. J. Appl. Physiol. 1987, 63, 359. 10. Weinstein-Lloyd, J.; Lee, J. H. J. Chem. Educ. 1995, 72, 1053. 11. Daly, D. J.; O’Sullivan, C. K.; Guilbault, G. G. Talanta 1999, 49, 667. 12. Ferapontova, E. E.; Grigorenko, V. G.; Egorov, A. M.; Borchers, T.; Ruzgas, T.; Gorton, L. Biosens. Bioelectron. 2001, 16, 147. 13. Toniolo, R.; Geatti, P.; Bontempeilli, G.; Schiavon, G. J. Electroanal. Chem. 2001, 514, 123. 14. Nozaki, O.; Kawamoto, H. Luminescence 2000, 15, 137. 15. Li, J.; Dasgupta, P. K. Anal. Chim. Acta. 2001, 442, 63. 16. Watson, C. A. Official and Standardized Methods of Analysis, 3rd ed.; The Royal Society of Chemistry: Cambridge, United Kingdom, 1994; pp 166–167.
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